Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants

Spheroids are a powerful tool for basic research and to reduce or replace in vivo (animal) studies but are not routinely banked nor shared. Here, we report the successful cryopreservation of hepatocyte spheroids using macromolecular (polyampholyte) cryoprotectants supplemented into dimethyl sulfoxide (DMSO) solutions. We demonstrate that a polyampholyte significantly increases post-thaw recovery, minimizes membrane damage related to cryo-injury, and remains in the extracellular space making it simple to remove post-thaw. In a model toxicology challenge, the thawed spheroids matched the performance of fresh spheroids. F-actin staining showed that DMSO-only cryopreserved samples had reduced actin polymerization, which the polyampholyte rescued, potentially linked to intracellular ice formation. This work may facilitate access to off-the-shelf and ready-to-use frozen spheroids, without the need for in-house culturing. Readily accessible 3-D cell models may also reduce the number of in vivo experiments.


Monolayer Cryomicroscopy Imaging
To further study intracellular ice formation, HepG2 cells were plated on coverslips at a density of 1 x 10 5 cells. 2 The coverslip was positioned on a quartz crucible with a cryoprotectant solution (5 mL) containing polyampholyte (40 mg.mL -1 ) and 10% DMSO, which was placed on a Linkam BCS 196 cryostage. To reach equilibrium, cells were incubated at 20°C for 10 min and subsequently frozen at 1 -5 °C.min -1 until -80 °C either with or without manual nucleation at -8°C using a cooling element. Following freezing, the cells were warmed to RT at 20°C.min -1 . Lynksis 32 software was used to edit and control the cryostage parameters and an Olympus CX41 microscope fitted with a UIS-20x/0.45//0/2FN22 lens and a Canon EOS 500D SLR digital camera was used for video recording. Image processing was completed using ImageJ v1.52 (National Institutes of Health, USA).

Spheroid Cryomicroscopy Imaging
Two studies were carried out with cryomicroscopy imaging to study the effects of intracellular ice formation of cells within spheroids in bulk freezing (i.e. suspension freezing) and surfacebased freezing. To the centre of a quartz crucible of 15 mm in diameter (Linkam Scientific Instruments, Salford), 100 µL of cell culture media supplemented with 40 mg.mL -1 of polyampholyte and 10% DMSO was added. Spheroids of 3000 cells were placed in the CPA solution and the top of the crucible was covered with a coverslip. The crucible was placed on a Linkam BCS 196 cryostage (Linkam Scientific Instruments, Salford), incubated at 20°C for 10 mins to achieve equilibrium and subsequently frozen at 1 °C.min -1 until -40 °C. To thaw the cells, the crucible was warmed to RT for 20 min. For surface-based freezing, spheroids were S3 positioned on a coverslip and placed on a quartz crucible containing 5 µL of cell culture media supplemented with 40 mg.mL -1 of polyampholyte and 10% DMSO. The spheroids were frozen using the Linkam cryostage as described above. Lynksis 32 software was used to edit and control the cryostage parameters and an Olympus CX41 microscope fitted with a UIS-20x/0.45//0/2FN22 lens and a Canon EOS 500D SLR digital camera was used for video recording. Image processing was completed using ImageJ v1.52 (National Institutes of Health, USA).

Effect of freezing on cytoskeleton:
Freeze/thaw spheroids, frozen in cryovials as a suspension with a cryoprotective agent (CPA) consisting of either MEM base media supplemented with 10% (v/v) FBS, 10% (v/v) DMSO and varying concentrations of polyampholyte (0 -80 mg.mL -1 ), were fixed with 2.5% paraformaldehyde for 30 min and then washed three times with PBS. After fixing, spheroids were permeabilized with 0.2% Triton X-100 in PBS for 10 min at RT and stained with Invitrogen ActinGreen TM 488 ReadyProbe TM reagent (0.145 mg.mL -1 in PBS) for 1 h.
Spheroids were also stained with a Hoechst 33342 nuclear stain (1 µg.mL -1 ) for 20 min and imaged using an Olympus FV3000 confocal microscope. Hoechst-stained nuclear material was excited using a diode laser with 350 nm and 461 nm emission wavelengths. The Alexa Fluor 488-Phalloidin-labeled actin filaments were excited and optically scanned using an argon-ion laser (excitation and emission wavelengths of 499 and 520 nm, respectively). 3 ImageJ was used to analyze the F-actin mean fluorescent intensity.

Polyampholyte Synthesis
Polyampholyte was synthesised as previously described. 1 Poly(methyl vinyl ether-alt-maleic anhydride) with an average Mn ≈ 80 kDa (10 g) was dissolved in THF (100 mL) heated to 50 °C. Dimethylamino ethanol (∼10 g) was added in excess. After 30 min, the waxy solid was dissolved in water (100 mL) and left to stir overnight. The remaining THF was removed under vacuum, and the resulting solution was purified in dialysis tubing (Spectra/Por, 12-14 kDa MWCO) for 72 h with 6 water changes. The polyampholyte was freeze dried, yielding an offwhite powder.
Dimethylamino ethanol (2 g) was added in excess forming a pink waxy solid. Following 30 min, the waxy solid was dissolved in water (50 mL) and left to stir overnight. After adding 50 mL of water, the reaction was stirred overnight before being purified by dialysis. The resulting solution was freeze-dried. The remaining THF was removed under vacuum, and the resulting solution was purified in dialysis tubing (Spectra/Por, 12-14 kDa MWCO) for 72 h with 6 water changes. The polyampholyte was freeze dried.

Intracellular Fluorescently Labelled Poly(ampholyte)s uptake
The intracellular uptake of polyampholytes was examined using fluorescent imaging with fluorescently labelled Polyampholytes dye. Briefly, 10-day-old HepG2 spheroids were